The long-range goal of this research is to define the precise sequence of biochemical events that follow a potentially mutagenic change introduced at a specific site in a biologically active DNA molecule by a reaction with a carcinogen and to determine whether or not this particular sequence of events leads to a tumor. Our current research has two immediate goals: (1) To develop a direct method for introducing a potentially mutagenic lesion into a single, predetermined site in biologically active DNA; (2) to develop a direct method for examining the biological expression of any given premutational lesion. We are interested in answering the following questions: (1) Which of the covalent modifications produced in DNA by reaction with a given carcinogen is mutagenic? (2) What kind of mutation does each different kind of premutational lesion in DNA produce? (3) How do different cellular repair systems handle different types of premutational lesions, and what role (if any) do these repair systems play in the mutation process? Our current studies employ the bacteriophage phi X174 as a source of biologically active DNA. We are introducing different potentially mutagenic lesions into single, preselected sites in gene G by a combination of chemical and enzymatic syntheses. The expression of these site-specific modifications is examined in vivo by transfection of spheroplasts carrying a functional copy of phi X gene G on a plasmid. This system is permissive for most, if not all, types of mutation in gene G. A similar, plasmid-bearing cell line is used to detect and propagate the mutant phage that results in the region of interest to define the precise nature of the mutation that has occurred in response to the original site-specific covalent modification. We believe that this new approach will provide a better understanding of the fundamental molecular mechanisms of mutagenesis by carcinogens. Hopefully it will also provide a more rational basis for establishing priorities for complicated and expensive testing of compounds that are potentially dangerous to man.